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Abstract

One of the main challenges in understanding the central nervous system is to measure the network dynamics of neuronal assemblies, while preserving the computational role of individual neurons. However, this is not possible with current techniques. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

Figures (5)

Random access second-harmonic generation (RASH) microscope. A fiber laser provided the excitation light, which comprised 200 fs width pulses at 80 MHz repetition rate. The laser beam was adjusted for optimal linear polarization via a half-wave (λ/2) plate. Beam passes were made through 45° AOM for angular spreading pre-compensation. A second half-wave (λ/2) plate was placed after the AOM to optimize the diffraction efficiencies of the 2 orthogonally mounted AODs (AOD-x and AOD-y). A scanning lens (SL) and a microscope tube lens (TL) expanded the beam before it was focused onto the specimen by the objective lens. The SHG signal was collected by an oil immersion condenser, band-pass filtered (BFP) and focalized by a collection lens (CL) into a GaAsP PMT.

Staining and imaging. a) A photograph of a sagittal cerebellar slice unstained, b) Cerebellar slice after incubation with FM4-64 (100 µg/ml) showing the dye loading throughout the tissue. c, d) Upper traces show sodium spikes typically recorded in the PC soma after current injection. Lower traces show calcium spikes coming from the dendritic compartment of the PC. These current clamp recording show normal physiological conditions in the unstained (c) and stained (d) condition. In these measurements, PCs were hyperpolarized at a holding potential of -70 mV. e) The four panels show traces of voltage transient (black line) in a single PC following stimulation of climbing fibers (green line) at different holding potentials (-60, -70, -80, -90 mV). For each holding potential, 20 sequential trials are shown superimposed. Red trace shows the average of the 20 trials. f) SHG image of cerebellar slice at three different depths of 10, 50 and 100 µm, oriented with the granule cell layer on the left and the molecular layer on the right. Examples of SHG signals from a PC (red arrow), granule cell (yellow arrow) and interneuron (blue arrow). The images were acquired with the same laser power across all three depths.

Optical recording of APs induced by afferent fibers stimulation. a) Traces of APs in a single PC following stimulation of climbing fibers (green line). 20 trials are shown superimposed. b) Simultaneous SHG recording of APs in 20 trials, corresponding to the electrophysiological trace in a). Integration time 100 µs, sampling frequency 2 kHz. c) The average of the 20 trials are presented for both electrical (red trace) and SHG (blue trace) signals, showing how well the SHG changes track the membrane potential. d) Electrical traces of 20 trials demonstrating jitter in the response of PCs to the afferent stimulation. e) Simultaneous recording of SHG in 20 trials showing the commensurate jitter in the optical recordings. Integration time 100 µs, sampling frequency 2 kHz. f) Averaged traces of both electrical and SHG signals, showing the effect of jitter on broadening and attenuation of APs when multiple trials are averaged. In these measurements, PCs were hyperpolarized at a holding potential of -70 mV. One second was allowed between the 20 individual multi-line scans.

Optical multi-unit recording of stimulated electrical activity. a) SHG image of a cerebellar slice taken at a depth of 90 µm. The multi-unit SHG recording was carried out from the lines drawn (dotted red) on the 5 PCs, with the integration time per membrane pass indicated. b) Multiplexed recording of APs from the 5 PCs following stimulation of climbing fibers (green lines). Each trace represents the average of 20 trials for each PCs. The time resolution is 0.47 ms. c) Superposition of SHG traces corrected for multiplex delay is shown. PC5 is not shown since it was quiescent.

Optical multi-unit recording of spontaneous electrical activity. a) SHG image from a cerebellar slice taken at a depth of 50 µm. The multi-unit SHG recording was carried out from the lines drawn (dotted red) on PC1 and PC2, with the integration time per membrane pass indicated. PC1 was also measured simultaneously by electrophysiology (shadow of electrode can be seen below PC1). b) SHG signal from PC1 showing a spontaneous AP recorded in a single trial. Each point represents 0.535 ms. Confidence intervals are drawn indicating the probability of the noise crossing thresholds. The probability of the noise crossing a threshold of 0.92 is 0.003%, indicating the event shown is an AP (see panel e). c) SHG signal from PC2, suggesting that no spontaneous activity was detected in this 150 ms sampling time. d) Simultaneous electrical recording of PC1, corresponding to the SHG trace shown in panel b. The electrical recording of spontaneous activity in PC1 before e) and after f) the SHG signal collection.